The long-term objective of this proposal is to investigate the biological development of chemical synapses with a multidisciplinary approach using physiological, morphological, immunocytochemical and genetic techniques. Our specific aims are to investigate (1) the entire developmental process of the synapse, following the stages from undifferentiated neuroblast to matured, functional synapse. (2) We will then investigate where and when de novo formation of synaptic vesicles takes place in developing neurons, (3) how extrinsic and intrinsic factors determine the de novo formation of synaptic vesicles, and (4) how the transmitter is packaged in the vesicle and how the vesicle is transported to the release site. We will also investigate (5) the development of other organelles specific to the presynaptic terminal, and (6) study the development of synaptic function in parallel with the morphological development of these organelles. We will then investigate (7) the conditions necessary for the maintenance of the established synapses. To do these experiments, we will use Drosophila neurons cultured in vitro. These cultures will enable us to follow the normal development of individual neurons from undifferentiated precursor cells or neuroblasts, to mature neurons with established synaptic contact. Additionally, by use of cultures from genetic mutants, we can perturb presynaptic membrane recycling and choline acetyltransferase synthesis and study the effects of these events on synaptic development. The results of the study will provide knowledge of the biological development of synapses and contribute to the understanding of developmental malfunctions of the nervous system and their resulting neurological disorders.